Antisense modulation of interferon gamma receptor 1 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of Interferon gamma receptor 1. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Interferon gamma receptor 1. Methods of using these compounds for modulation of Interferon gamma receptor 1 expression and for treatment of diseases associated with expression of Interferon gamma receptor 1 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of Interferon gamma receptor 1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Interferon gamma receptor 1. Such compounds have been shown to modulate the expression of Interferon gamma receptor 1.

BACKGROUND OF THE INVENTION

[0002] Cytokines are small to medium sized proteins and glycoproteins that mediate highly potent biological effects on many cell types in networks or cascades. They have critical roles in hematopoiesis, inflammatory responses and the development and maintenance of immune responses. Typical properties in networks of cytokines are pleiotropy, redundancy, synergistic activity and antagonistic effects upon each other (Townsend and McKenzie, J. Cell Sci., 2000, 113, 3549-3550). Knowledge of how cytokine networks are comprised and operate is important in understanding how they mediate their diverse effects on biological systems (Townsend and McKenzie, J. Cell Sci., 2000, 113, 3549-3550).

[0003] Interferon gamma (IFN-gamma) is a cytokine secreted by activated T lymphocytes and natural killer (NK) cells. It is involved in the modulation of a wide variety of immunological and inflammatory responses including activation of macrophages, cytotoxic T cells and Natural Killer (NK) cells, regulation of antibody production by B lymphocytes and control of apoptosis (Billiau, Adv. Immunol., 1996, 62, 61-130).

[0004] Interferon gamma receptor complexes are expressed on almost all nucleated cells and show species specificity in their ability to bind interferon gamma (Dorman and Holland, Cytokine Growth Factor Rev., 2000, 11, 321-333). The functional interferon gamma receptor is composed of two 90 kDa polypeptides designated interferon gamma receptor 1 (also known as IFNGR1, IFN-gamma receptor-alpha chain and CD119w) and interferon gamma receptor 2 (also known as IFNGR2, IFN-gamma receptor-beta, IFN-gamma transducer 1, AF-1 and GAF).

[0005] The extracellular portion of interferon gamma receptor 1 contains the IFN-gamma ligand-binding domain and the intracellular portion contains the domains necessary for signal transduction and receptor recycling (Dorman and Holland, Cytokine Growth Factor Rev., 2000, 11, 321-333). In the absence of stimulation, interferon gamma receptors 1 and 2 are not strongly associated with each other. Upon binding of IFN-gamma as a homodimer to the two interferon gamma receptor 1 proteins, interferon gamma receptors 1 and 2, and their constitutively-associated Janus kinases (JAK1 and JAK2, respectively) are brought into proximity so that the signaling cascade can begin via phosphorylation of tyrosine-440 on interferon gamma receptor 1 (Dorman and Holland, Cytokine Growth Factor Rev., 2000, 11, 321-333). Signals are transmitted to the nucleus via the Jak-STAT mechanism of signal transduction involving a cascade of tyrosine phosphorylations of STAT proteins (signal transducer and activator of transcription) in the cytoplasm.

[0006] Human interferon gamma receptor 1 has been mapped to chromosome 6q23-6q24, a region which undergoes rearrangements in ovarian carcinoma and malignant melanoma (Le Coniat et al., Hum. Genet., 1989, 84, 92-94). In addition, partial deletions and translocations involving bands 6q14 to 6q27 are frequently observed in various types of lymphoid cell malignancies, leukemias and lymphomas (Le Coniat et al., Hum. Genet., 1989, 84, 92-94).

[0007] Complete absence of human IFN-gamma responsiveness due to a mutation in either interferon gamma receptor 1 or 2 is typically associated with severe mycobacterial infections in lungs, viscera, lymph nodes, blood and bone marrow (Dorman and Holland, Cytokine Growth Factor Rev., 2000, 11, 321-333). A clinical phenotype associated with autosomal dominant mutations of interferon gamma receptor 1 (leading to partial interferon gamma receptor 1 deficiency) is milder than seen in individuals with complete absence of INF-gamma responsiveness and infections are usually responsive to appropriate antimicrobial therapy (Dorman and Holland, Cytokine Growth Factor Rev., 2000, 11, 321-333.).

[0008] The expression of the human interferon gamma receptor complex is modulated by environmental signals in human malignant T cells and thymocytes (Novelli et al., J. Immunol., 1994, 152, 496-504). The upregulation of IFN-gamma expression promotes T cell proliferation in T cells with low levels of interferon gamma receptor complex and apoptosis in T cells with high levels of interferon gamma receptor complex (Novelli et al., J. Immunol., 1994, 152, 496-504).

[0009] Although IFN-gamma signaling typically exerts antiviral effects, in some cases of bacterial infections, endogenous IFN-gamma can act as a detriment to the host. In the case of HIV infection, activation of monocytoid cells by IFN-gamma signaling was found to stimulate rather than inhibit virus replication (Biswas et al., J. Exp. Med., 1992, 176, 739-750).

[0010] In addition, there are conflicting reports on the effects of IFN-gamma signaling on tumor growth in mice. For example, investigators have inserted the IFN-gamma gene into non-immunogenic murine tumor cells with high metastasizing potential and found that the IFN-gamma secreting cells had less ability to develop tumors than the parental cells (Gansbacher et al., Cancer Res., 1990, 50, 7820-7825). On the other hand, a metastasizing murine mammary carcinoma productively transfected with the IFN-gamma gene was found to metastasize more extensively than the untransfected tumor line (Ferrantini et al., J. Immunol., 1994, 153, 4604-4615).

[0011] In experimental animal models of autoimmune disease including: autoimmune thyroiditis, experimental autoimmune peripheral neuritis and autoimmune insulin-dependent diabetes, experimental autoimmune encephalomyelitis, autoimmune arthritis and autoimmune insulinitis, IFN-gamma was either found to facilitate induction of disease or to aggravate disease manifestations (Billiau, Adv. Immunol., 1996, 62, 61-130). Increased levels of IFN-gamma mRNA have been-observed in intestinal propria lamina of Crohn's disease patients (Fais et al., J. Interferon Res., 1994, 14, 235-238).

[0012] Deregulation of IFN-gamma signaling has been implicated in autoimmune disease, complications due to viral infections and cancer. Furthermore, deregulated apoptosis signaling may impinge on other age-related disorders such as osteoporosis and atherosclerosis. Modulation of expression of components of the interferon gamma receptor complex, including interferon gamma receptor 1 may prove to be a useful strategy with which to down-regulate IFN-gamma signaling in cases where excessive signaling precipitates disease.

[0013] Attempts to modulate the process of IFN-gamma signaling have thus far been essentially limited to inhibition of IFN-gamma itself. Strategies aimed at inhibition of interferon gamma receptor 1 function are as yet untested as investigative or therapeutic protocols. Consequently there remains a long felt need for agents capable of effectively inhibiting interferon gamma receptor 1 function.

[0014] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of interferon gamma receptor 1 expression.

[0015] The present invention provides compositions and methods for modulating interferon gamma receptor 1 expression.

SUMMARY OF THE INVENTION

[0016] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Interferon gamma receptor 1, and which modulate the expression of Interferon gamma receptor 1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Interferon gamma receptor 1 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Interferon gamma receptor 1 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Interferon gamma receptor 1, ultimately modulating the amount of Interferon gamma receptor 1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Interferon gamma receptor 1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Interferon gamma receptor 1” encompass DNA encoding Interferon gamma receptor 1, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of Interferon gamma receptor 1. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

[0018] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding Interferon gamma receptor 1. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Interferon gamma receptor 1, regardless of the sequence(s) of such codons.

[0019] It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.

[0020] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0021] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0022] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0023] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0024] Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites.

[0025] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

[0026] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0027] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0028] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0029] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0030] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0031] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

[0032] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0033] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0034] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a ₃′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0035] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0036] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0037] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0038] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include-, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0039] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0040] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N— alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0041] A further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0042] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2 ′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2¹-O—CH₂—CH═CH₂) and 2′-fluoro (2′F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0043] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido [4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0044] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0045] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)−(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0046] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0047] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0048] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0049] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0050] The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.

[0051] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0052] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0053] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0054] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0055] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid.- Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

[0056] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0057] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Interferon gamma receptor 1 is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

[0058] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Interferon gamma receptor 1, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding Interferon gamma receptor 1 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Interferon gamma receptor 1 in a sample may also be prepared.

[0059] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0060] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U. S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

[0061] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Prefered bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Prefered fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also prefered are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly prefered combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety.

[0062] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0063] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0064] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0065] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0066] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

[0067] Emulsions

[0068] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a,w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.

[0069] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0070] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0071] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0072] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0073] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0074] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0075] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0076] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0077] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0078] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0079] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci. , 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

[0080] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0081] Liposomes

[0082] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0083] Liposomes are unilamellar or multilamellat vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0084] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0085] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0086] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0087] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0088] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

[0089] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0090] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0091] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0092] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0093] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0094] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more-specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0095] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

[0096] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0097] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

[0098] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0099] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0100] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0101] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0102] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0103] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0104] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0105] Penetration Enhancers

[0106] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0107] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0108] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0109] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0110] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0111] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0112] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0113] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

[0114] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

[0115] Carriers

[0116] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0117] Excipients

[0118] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0119] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0120] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0121] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0122] Other Components

[0123] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0124] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0125] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more-antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0126] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0127] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0128] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0129] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy amidites

[0130] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 21-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.

[0131] Oligonucleotides containing 5-methyl-2¹-deoxycytidine (5-Me-C) nucleotides were synthesized according to published methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

[0132] 2′-Fluoro amidites

[0133] 2′-Fluorodeoxyadenosine amidites

[0134] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a 2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0135] 2′-Fluorodeoxyguanosine

[0136] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

[0137] 2′ -Fluorouridine

[0138] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

[0139] 2′-Fluorodeoxycytidine

[0140] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

[0141] 2′-O-(2-Methoxyethyl) modified amidites

[0142] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.

[0143]2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0144] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.).

[0145] 2′-O-Methoxyethyl-5-methyluridine

[0146] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions.

[0147] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0148] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂SO4, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

[0149] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0150] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.

[0151] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0152] A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃ was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

[0153] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0154] A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxy-trityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH₄0H (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH₃ gas was added and the vessel heated to 100° C. for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

[0155] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine

[0156] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl₃ (700 mL) *and extracted with saturated NaHCO₃ (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

[0157] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine-3′-amidite

[0158] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (74 g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra(isopropyl)-phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂Cl₂ (300 mL), and the extracts were combined, dried over MgSO₄ and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

[0159] 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites

[0160] 2′-(Dimethylaminooxyethoxy) nucleoside amidites

[0161] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.

[0162] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0163] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to −10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product.

[0164] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0165] In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure<100 psig). The reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. [Alternatively, once. the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product.

[0166] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0167] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop. After the addition was complete, the reaction was stirred for 4 hrs. By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%).

[0168] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0169] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate was washed with ice cold CH₂Cl₂ and the combined organic phase was washed with water, brine and dried over anhydrous Na₂SO₄. The solution was concentrated to get 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was strirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%).

[0170] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0171] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in a solution of 1 M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was added and extracted with ethyl acetate (2×25 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄ and evaporated to dryness The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH₂Cl₂ to get 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).

[0172] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0173] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).

[0174] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0175] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P₂O₅ under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained was dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a few drops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

[0176] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0177] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P₂O₅ under high vacuum overnight at 40° C. Then the reaction mixture was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrous Na₂SO₄ and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%).

[0178] 2′-(Aminooxyethoxy) nucleoside amidites

[0179] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.

[0180] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0181] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0182] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0183] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.

[0184] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0185] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O²—, 2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155° C. for 26 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3×200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid.

[0186] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl uridine

[0187] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃ solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine) gives the title compound.

[0188] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0189] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. The reaction mixture is stirred overnight and the solvent evaporated. The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound.

Example 2

[0190] Oligonucleotide Synthesis

[0191] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.

[0192] Phosphorothioates (P═S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0193] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0194] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or U.S. Pat. No. 5,625,050, herein incorporated by reference.

[0195] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0196] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0197] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0198] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0199] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

[0200] Oligonucleoside Synthesis

[0201] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0202] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0203] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0204] PNA Synthesis

[0205] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5

[0206] Synthesis of Chimeric Oligonucleotides

[0207] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0208] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0209] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2′-O-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2′ positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to ½ volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0210] [2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0211] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0212] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxy Phosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0213] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxy phosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0214] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0215] Oligonucleotide Isolation

[0216] After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by ³¹p nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0217] Oligonucleotide Synthesis—96 Well Plate Format

[0218] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0219] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0220] Oligonucleotide Analysis—96 Well Plate Format

[0221] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/ACEs MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0222] Cell Culture and Oligonucleotide Treatment

[0223] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following 4 cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, Ribonuclease protection assays, or RT-PCR.

[0224] T-24 Cells:

[0225] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0226] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0227] A549 Cells:

[0228] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0229] NHDF Cells:

[0230] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0231] HEK Cells:

[0232] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0233] Treatment with Antisense Compounds:

[0234] When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Gibco BRL) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0235] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments.

Example 10

[0236] Analysis of Oligonucleotide Inhibition of Interferon Gamma Receptor 1 Expression

[0237] Antisense modulation of Interferon gamma receptor 1 expression can be assayed in a variety of ways known in the art. For example, Interferon gamma receptor 1 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMS 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0238] Protein levels of Interferon gamma receptor 1 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to Interferon gamma receptor 1 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0239] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0240] Poly(A)+ mRNA Isolation

[0241] Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred-to a fresh 96-well plate.

[0242] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12

[0243] Total RNA Isolation

[0244] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.

[0245] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0246] Real-Time Quantitative PCR Analysis of Interferon Gamma Receptor 1 mRNA Levels

[0247] Quantitation of Interferon gamma receptor 1 mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE, FAM, or VIC, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0248] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0249] PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0250] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, Analytical Biochemistry, 1998, 265, 368-374.

[0251] In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.

[0252] Probes and primers to human Interferon gamma receptor 1 were designed to hybridize to a human Interferon gamma receptor 1 sequence, using published sequence information (GenBank accession number NM_(—)000416, incorporated herein as SEQ ID NO:3). For human Interferon gamma receptor 1 the PCR primers were: forward primer: CTTAGAAAAGGAGGTGGTCTGTGAA (SEQ ID NO: 4) reverse primer: CCTGGATTGTCTTCGGTATGC (SEQ ID NO: 5) and the PCR probe was: FAM-CGTTGTCTCCAGCAACAGTTCCAGGC-TAMRA (SEQ ID NO: 6)

[0253] where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9)

[0254] where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14

[0255] Northern Blot Analysis of Interferon Gamma Receptor 1 mRNA Levels

[0256] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0257] To detect human Interferon gamma receptor 1, a human Interferon gamma receptor 1 specific probe was prepared by PCR using the forward primer CTTAGAAAAGGAGGTGGTCTGTGAA (SEQ ID NO: 4) and the reverse primer CCTGGATTGTCTTCGGTATGC (SEQ ID NO: 5). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0258] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0259] Antisense Inhibition of Human Interferon Gamma Receptor 1 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0260] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Interferon gamma receptor 1 RNA, using published sequences (GenBank accession number NM_(—)000416, incorporated herein as SEQ ID NO: 3, and the complement of residues 59001-85000 of GenBank accession number AL050337 which is incorporated herein as SEQ ID NO: 10). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human Interferon gamma receptor 1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human Interferon gamma receptor 1 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO 133747 Coding 3 186 tggcatgatctggtactccc 95 11 133782 Coding 3 1120 ccacgtcaggaatattttct 79 12 133783 Coding 3 1156 tctctctctctattggagtc 69 13 133785 Coding 3 1204 aagcgatgctgccaggttca 0 14 133794 Coding 3 1490 gaaaattctttggaatcttc 0 15 133795 Stop 3 1502 agctgatctcatgaaaattc 79 16 Codon 133799 3′UTR 3 1572 tcataatcttttcatgaaat 60 17 133800 3′UTR 3 1579 tctgagatcataatcttttc 92 18 133802 3′UTR 3 1642 cctctttacgctttcatgta 4 19 133818 Intron 10 15268 ccctgccccagagatttgtg 11 20 2 147599 Coding 3 89 gcggtgcccatctcagccct 1 21 147600 Coding 3 98 cccagatccgcggtgcccat 92 22 147601 Coding 3 125 ttagttggtgtaggcactga 0 23 147602 Coding 3 128 acattagttggtgtaggcac 84 24 147603 Coding 3 148 tgttataggattcaattgta 70 25 147604 Coding 3 163 atacgatagggttcatgtta 0 26 147605 Coding 3 174 gtactcccaatatacgatag 14 27 147606 Coding 3 177 ctggtactcccaatatacga 85 28 147607 Coding 3 180 gatctggtactcccaatata 77 29 147608 Coding 3 193 ggacctgtggcatgatctgg 79 30 147609 Coding 3 306 tggatcaccaacatgatcag 0 31 147610 Coding 3 340 ccctggctttaactctgacc 0 32 147611 Coding 3 344 ccaaccctggctttaactct 1 33 147612 Coding 3 347 tgtccaaccctggctttaac 40 34 147613 Coding 3 370 actttgcataggcagattct 0 35 147614 Coding 3 373 ctgactttgcataggcagat 0 36 147615 Coding 3 387 tacagcaaattcttctgact 9 37 147616 Coding 3 420 cagtttaggtggtccaattt 14 38 147617 Coding 3 428 ctgatatccagtttaggtgg 0 39 147618 Coding 3 450 catgatttgcttctcctcct 0 40 147619 Coding 3 489 gtctccatttacaaaaactg 67 41 147620 Coding 3 498 ttcctgctcgtctccattta 0 42 147621 Coding 3 505 aatcgacttcctgctcgtct 0 43 147622 Coding 3 530 atgtaacaggtagtttcggg 65 44 147623 Coding 3 554 ctcacatacacattgtacac 69 45 147624 Coding 3 559 tcattctcacatacacattg 11 46 147625 Coding 3 565 ttccgttcattctcacatac 0 47 147626 Coding 3 592 gcgtgagtattttatactgg 77 48 147627 Coding 3 594 ctgcgtgagtattttatact 12 49 147628 Coding 3 609 acaatcatcttccttctgcg 0 50 147629 Coding 3 611 tcacaatcatcttccttctg 0 51 147630 Coding 3 623 cactgaatctcgtcacaatc 49 52 147631 Coding 3 628 actggcactgaatctcgtca 85 53 147632 Coding 3 662 tactgagaattcagtgagga 0 54 147633 Coding 3 665 cagtactgagaattcagtga 58 55 147634 Coding 3 670 aaacacagtactgagaattc 2 56 147635 Coding 3 679 cttctgctgaaacacagtac 67 57 147636 Coding 3 698 ccccacacatgtaagactcc 0 58 147637 Coding 3 702 aacaccccacacatgtaaga 68 59 147638 Coding 3 757 cttttatactgctattgaaa 0 60 147639 Coding 3 785 gcagcaacaactggaatcca 83 61 147640 Coding 3 793 gtagtaaagcagcaacaact 5 62 147641 Coding 3 809 ctaagcactagaaagagtag 79 63 147642 Coding 3 912 taaagtagcacttcttacca 42 64 147643 Coding 3 923 ggttttgtctctaaagtagc 64 65 147644 Coding 3 964 atggctggtatgacgtgatg 71 66 147645 Coding 3 1013 gttgctggagacaacggctc 76 67 147646 Coding 3 1017 aactgttgctggagacaacg 85 68 147647 Coding 3 1057 gttccacttttcctggattg 0 69 147648 Coding 3 1070 agttcttctgtatgttccac 86 70 147649 Coding 3 1072 aaagttcttctgtatgttcc 71 71 147650 Coding 3 1135 gatggctgcccgggaccacg 0 72 147651 Coding 3 1138 tcagatggctgcccgggacc 83 73 147652 Coding 3 1163 gaagaactctctctctctat 0 74 147653 Coding 3 1175 ctacttaaaggtgaagaact 0 75 147654 Coding 3 1183 actggttactacttaaaggt 5 76 147655 Coding 3 1220 gagtgatacgagtttaaagc 55 77 147656 Coding 3 1247 gagtgatcactctcagaaca 29 78 147657 Coding 3 1251 tctggagtgatcactctcag 78 79 147658 Coding 3 1293 gctatgtgattccagacagc 0 80 147659 Coding 3 1316 ggaaattctgagtcagataa 0 81 147660 Coding 3 1369 ttacggttatgagctcttgt 10 82 147661 Coding 3 1400 ttatcataaccaaaggaggt 49 83 147662 Coding 3 1435 tatcatccacaagtagatcc 41 84 147663 Coding 3 1439 ccgctatcatccacaagtag 0 85 147664 Coding 3 1446 ctctttaccgctatcatcca 63 86 147665 Coding 3 1451 aaggactctttaccgctatc 87 87 147666 Coding 3 1474 cttctgttggtctataacca 0 88

[0261] As shown in Table 1, SEQ ID NOs 11, 12, 13, 16, 17, 18, 22, 24, 25, 28, 29, 30, 41, 44, 45, 48, 53, 55, 57, 59, 61, 63, 65, 66, 67, 68, 70, 71, 73, 77, 79, 86 and 87 demonstrated at least 50% inhibition of human Interferon gamma receptor 1 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.

Example 16

[0262] Western Blot Analysis of Interferon Gamma Receptor 1 Protein Levels

[0263] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Interferon gamma receptor 1 is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 88 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2 atgcattctg cccccaagga 20 3 2059 DNA Homo sapiens CDS (44)...(1513) 3 ccgcaggcgc tcggggttgg agccagcgac cgtcggtagc agc atg gct ctc ctc 55 Met Ala Leu Leu 1 ttt ctc cta ccc ctt gtc atg cag ggt gtg agc agg gct gag atg ggc 103 Phe Leu Leu Pro Leu Val Met Gln Gly Val Ser Arg Ala Glu Met Gly 5 10 15 20 acc gcg gat ctg ggg ccg tcc tca gtg cct aca cca act aat gtt aca 151 Thr Ala Asp Leu Gly Pro Ser Ser Val Pro Thr Pro Thr Asn Val Thr 25 30 35 att gaa tcc tat aac atg aac cct atc gta tat tgg gag tac cag atc 199 Ile Glu Ser Tyr Asn Met Asn Pro Ile Val Tyr Trp Glu Tyr Gln Ile 40 45 50 atg cca cag gtc cct gtt ttt acc gta gag gta aag aac tat ggt gtt 247 Met Pro Gln Val Pro Val Phe Thr Val Glu Val Lys Asn Tyr Gly Val 55 60 65 aag aat tca gaa tgg att gat gcc tgc atc aat att tct cat cat tat 295 Lys Asn Ser Glu Trp Ile Asp Ala Cys Ile Asn Ile Ser His His Tyr 70 75 80 tgt aat att tct gat cat gtt ggt gat cca tca aat tct ctt tgg gtc 343 Cys Asn Ile Ser Asp His Val Gly Asp Pro Ser Asn Ser Leu Trp Val 85 90 95 100 aga gtt aaa gcc agg gtt gga caa aaa gaa tct gcc tat gca aag tca 391 Arg Val Lys Ala Arg Val Gly Gln Lys Glu Ser Ala Tyr Ala Lys Ser 105 110 115 gaa gaa ttt gct gta tgc cga gat gga aaa att gga cca cct aaa ctg 439 Glu Glu Phe Ala Val Cys Arg Asp Gly Lys Ile Gly Pro Pro Lys Leu 120 125 130 gat atc aga aag gag gag aag caa atc atg att gac ata ttt cac cct 487 Asp Ile Arg Lys Glu Glu Lys Gln Ile Met Ile Asp Ile Phe His Pro 135 140 145 tca gtt ttt gta aat gga gac gag cag gaa gtc gat tat gat ccc gaa 535 Ser Val Phe Val Asn Gly Asp Glu Gln Glu Val Asp Tyr Asp Pro Glu 150 155 160 act acc tgt tac att agg gtg tac aat gtg tat gtg aga atg aac gga 583 Thr Thr Cys Tyr Ile Arg Val Tyr Asn Val Tyr Val Arg Met Asn Gly 165 170 175 180 agt gag atc cag tat aaa ata ctc acg cag aag gaa gat gat tgt gac 631 Ser Glu Ile Gln Tyr Lys Ile Leu Thr Gln Lys Glu Asp Asp Cys Asp 185 190 195 gag att cag tgc cag tta gcg att cca gta tcc tca ctg aat tct cag 679 Glu Ile Gln Cys Gln Leu Ala Ile Pro Val Ser Ser Leu Asn Ser Gln 200 205 210 tac tgt gtt tca gca gaa gga gtc tta cat gtg tgg ggt gtt aca act 727 Tyr Cys Val Ser Ala Glu Gly Val Leu His Val Trp Gly Val Thr Thr 215 220 225 gaa aag tca aaa gaa gtt tgt att acc att ttc aat agc agt ata aaa 775 Glu Lys Ser Lys Glu Val Cys Ile Thr Ile Phe Asn Ser Ser Ile Lys 230 235 240 ggt tct ctt tgg att cca gtt gtt gct gct tta cta ctc ttt cta gtg 823 Gly Ser Leu Trp Ile Pro Val Val Ala Ala Leu Leu Leu Phe Leu Val 245 250 255 260 ctt agc ctg gta ttc atc tgt ttt tat att aag aaa att aat cca ttg 871 Leu Ser Leu Val Phe Ile Cys Phe Tyr Ile Lys Lys Ile Asn Pro Leu 265 270 275 aag gaa aaa agc ata ata tta ccc aag tcc ttg atc tct gtg gta aga 919 Lys Glu Lys Ser Ile Ile Leu Pro Lys Ser Leu Ile Ser Val Val Arg 280 285 290 agt gct act tta gag aca aaa cct gaa tca aaa tat gta tca ctc atc 967 Ser Ala Thr Leu Glu Thr Lys Pro Glu Ser Lys Tyr Val Ser Leu Ile 295 300 305 acg tca tac cag cca ttt tcc tta gaa aag gag gtg gtc tgt gaa gag 1015 Thr Ser Tyr Gln Pro Phe Ser Leu Glu Lys Glu Val Val Cys Glu Glu 310 315 320 ccg ttg tct cca gca aca gtt cca ggc atg cat acc gaa gac aat cca 1063 Pro Leu Ser Pro Ala Thr Val Pro Gly Met His Thr Glu Asp Asn Pro 325 330 335 340 gga aaa gtg gaa cat aca gaa gaa ctt tct agt ata aca gaa gtg gtg 1111 Gly Lys Val Glu His Thr Glu Glu Leu Ser Ser Ile Thr Glu Val Val 345 350 355 act act gaa gaa aat att cct gac gtg gtc ccg ggc agc cat ctg act 1159 Thr Thr Glu Glu Asn Ile Pro Asp Val Val Pro Gly Ser His Leu Thr 360 365 370 cca ata gag aga gag agt tct tca cct tta agt agt aac cag tct gaa 1207 Pro Ile Glu Arg Glu Ser Ser Ser Pro Leu Ser Ser Asn Gln Ser Glu 375 380 385 cct ggc agc atc gct tta aac tcg tat cac tcc aga aat tgt tct gag 1255 Pro Gly Ser Ile Ala Leu Asn Ser Tyr His Ser Arg Asn Cys Ser Glu 390 395 400 agt gat cac tcc aga aat ggt ttt gat act gat tcc agc tgt ctg gaa 1303 Ser Asp His Ser Arg Asn Gly Phe Asp Thr Asp Ser Ser Cys Leu Glu 405 410 415 420 tca cat agc tcc tta tct gac tca gaa ttt ccc cca aat aat aaa ggt 1351 Ser His Ser Ser Leu Ser Asp Ser Glu Phe Pro Pro Asn Asn Lys Gly 425 430 435 gaa ata aaa aca gaa gga caa gag ctc ata acc gta ata aaa gcc ccc 1399 Glu Ile Lys Thr Glu Gly Gln Glu Leu Ile Thr Val Ile Lys Ala Pro 440 445 450 acc tcc ttt ggt tat gat aaa cca cat gtg cta gtg gat cta ctt gtg 1447 Thr Ser Phe Gly Tyr Asp Lys Pro His Val Leu Val Asp Leu Leu Val 455 460 465 gat gat agc ggt aaa gag tcc ttg att ggt tat aga cca aca gaa gat 1495 Asp Asp Ser Gly Lys Glu Ser Leu Ile Gly Tyr Arg Pro Thr Glu Asp 470 475 480 tcc aaa gaa ttt tca tga gatcagctaa gttgcaccaa ctttgaagtc 1543 Ser Lys Glu Phe Ser * 485 tgattttcct ggacagtttt ctgctttaat ttcatgaaaa gattatgatc tcagaaattg 1603 tatcttagtt ggtatcaacc aaatggagtg acttagtgta catgaaagcg taaagaggat 1663 gtgtggcatt ttcacttttg gcttgtaaag tacagacttt tttttttttt taaacaaaaa 1723 aagcattgta acttatgaac ctttacatcc agataggtta ccagtaacgg aacatatcca 1783 gtactcctgg ttcctaggtg agcaggtgat gccccaggga cctttgtagc cacttcactt 1843 tttttctttt ctctgccttg gtatagcata tgtgttttgt aagtttatgc atacagtaat 1903 tttaagtaat ttcagaagaa attctcgaag cttttcaaaa ttggacttaa aatctaattc 1963 aaactaatag aattaatgga atatgtaaat agaaacgtgt atatttttta tgaaacatta 2023 cagttagaga tttttaaata aagaatttta aaactc 2059 4 25 DNA Artificial Sequence PCR Primer 4 cttagaaaag gaggtggtct gtgaa 25 5 21 DNA Artificial Sequence PCR Primer 5 cctggattgt cttcggtatg c 21 6 26 DNA Artificial Sequence PCR Probe 6 cgttgtctcc agcaacagtt ccaggc 26 7 19 DNA Artificial Sequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNA Artificial Sequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20 DNA Artificial Sequence PCR Probe 9 caagcttccc gttctcagcc 20 10 26000 DNA Homo sapiens 10 ctctaaattc ttatcagtga cccaattata gcttagccag ccataaattc aactatacct 60 gcttcagtcc cacacctctt tacaagcttg ttttgttcat catatattta tttaataagt 120 ggccacagac attggttcag ctgctcaatg ctgacagtga tgattcagga ttacatacaa 180 ttgatgattc aggatcatca atattccata aatattaatg gagtaatagg cacttggcta 240 ggtgctcggg gtctgacagt gaagaaaaca aatttatttt ttagaggaag agggagacta 300 ctgaaacaaa attttctacg tgtgtatata catattttat gtatgcacac acacacggat 360 gcatttcatt aaatctcaga ttaagtccct gtttacgcac ctataatcgt gttgctctcc 420 ttggtaacaa ttactacaat ttcagtaatt atttgtgaaa tatatatata tataaaatgc 480 atatatttaa cacatacaat ttattcatat ttaattcaaa tttaatagat atattaaatt 540 tccagtagaa tctccagata gtagtaagta ctatggaata aacaaagtta aataagggcc 600 agtgtaaagg catgggtaga gtgtgagagg gcaagagtat tggctataat ttacagacta 660 atcagcggaa tttaaactat atgcttcagt tacaaatctt cacgctgtct cctttaaagg 720 gaagctggca gacatttctg ctttggagaa aagaggaagg aaatgcactg tgctcagagc 780 caaaatgcac agtttgttcc acacacacag gctctgttct cattggatca tatttctagc 840 ttcttaaatt gttttatttg gcctattaac ttgaaaggca ggtgagtatg gtcttcaaaa 900 tgtagccttg ctcctgagca gcagcacttc agtatacact tggctttgga atgatctacg 960 ctccacccca atttgttttc ttcattttaa aaactaatct gaggctctag tggggaccaa 1020 cgtatacttg gatattcccc agccattagg cattggaaat ttatcaagga aagctggcac 1080 cgctcatttg tttctgtgtt agctgtatga gatttttata tgggatttta aaatacagtt 1140 tattgctctt cttcctacct ctcttattag ccggtggcca cccccatctt ttaactttat 1200 tcaggctaat tttaccttat ccttcaggtc acaacataat ggtaacttcc ttaaaaaaga 1260 atttccctaa atccagacac agtggctcac acctgtaatc ccagcatttt gggagcctga 1320 ggcaggagaa tcatttgagc ccaggagttt gagaccagcc tgagcaacac agcaagaccc 1380 cagctctata aaaaatttaa aaactagccg ggcataaggc caggcgaggt ggctcactcc 1440 tgtaatccca gcactttggg aggccaaggt gggcggattg cttgagctca ggagtttgag 1500 atcagcctgg gcaacatggt gaaaccccat ctccacaaaa gacacaaaaa aatgcctgtg 1560 gtcccagcta cttgggaggc tgaggtggaa ggatggcttg cgcccgggag gtggaggttg 1620 cagtcagctg agattgtgcc actgtacctc agctaggtga tacagtcaga ccctgtctca 1680 aaaaaaaaaa aaaaaattag ccaggcatgg tggtgtacac ctgtagtccc agctatttgg 1740 gaggctgggg caagaggatc atttgggccc aggagtttga ggctgcagta agctatgatc 1800 atgcctctgc actgcagcct agacgacaga gtgagaccct gtctcaaaag aaagaaaaaa 1860 gaaaagaaag actttcttta aaatgtagac taagttaaat tcctgtttat actcctttaa 1920 tctcttccca ttgctctcct tgggaatgat gatcacgatt tcagtacttg tgtaattcct 1980 tgtttaacag ctatctccct cagtagacag taagctccgt gagggcaggg gccatgtctg 2040 tcttgctcag gtctttatct ctgtgcctgg tcatgtcagt cacagacagt atcacttgat 2100 aatattcacc caattaataa atatatttag tctccaaatg actaactagc aaagtcttca 2160 gaaggatacg tagggtggtg ggtgctgtga ttgtgaaact tgcccagatc cctctcagga 2220 acagaagact gggttctgtc cccttcctga tgctaggagg gctgccagta gacaccctca 2280 accgtcagcg ttcctctggg gaatgcctcc atgaaagaaa tggacagccc caaaatcatg 2340 acagttggta ttcaatgact aatcagtgct gggctttaag acccagcccc tcataccaac 2400 tcaagacacc tttacaaggc catcccaact ccagagcgcc ccttgggagc ctttactggg 2460 atggcatcac agatcaactt cttcctccgc tctgcttcct ccacctcccc ttcacagacg 2520 tttattccaa gagcactccc taatcaatca atgccttata ccttaatctc gacctcagag 2580 tttactgcca gggtaaccca gcctgtgaca agtaccacaa aaatgtttac aaactggata 2640 tcatattttg tcctactcca tagacgccta aacggtaaag atcagttatt tatccttgca 2700 tccttacaac gactagaagg tgtctttcat atagtggaca tcagaaacac atcaaaggaa 2760 atattctccc taaacttagt ctagattgag ctaaattctt ctgattcaaa gtgtggtcct 2820 gttgaaaggg gcaccagcat cccctgtgca ttttgtccga aatacaaaat ctcaagaccc 2880 aacctgaatt agaactactg aatccaaatc tgcattttat caagatcccc agatggttca 2940 catgtatgtt gaagtttgag aagtactgag ctaagagatt tattaataca agtaaaatga 3000 tctggctgat aatacctcta gtttatatag tgtctttata aggattttcc ctctttaaaa 3060 attttcctta acactagtaa tcatgcagtc tttcactcaa caaatactaa ttgagtgcca 3120 agtaaagatg tcagctctac ggagcttaca ttttagaagg agaagaaaac aaatatcaag 3180 gtgtatatta tagtgtacag caaggtgaaa agtgccatga aaaaacagta gggcggggta 3240 aggaggagga gaaggtgtgt tgcaatttaa aatttttaac aaaagttcat gatactcaga 3300 gaattgaaac aggtgagatc attagacatt cgcatgtttg agcacaagcg ctgaaggact 3360 tagcaatgtg gctgcagtgg aataggagag atgcaaaata acagacaaac ccagagaggt 3420 aagagagcag acctcttcat gagaggctgc ctgataaact gatttgacac tgaattgctg 3480 agaaggtaat gcaaatagtt tttttctcca agaacaaagt agttcttggt caagccgatt 3540 tgatttgggc tgtgggaatc tgcacaaacc atttcactag ctaagtctca ggttcctcaa 3600 atgaaaaagc agggactgga aatggctttt gaagtctcat tctcttctaa aaatgtcagg 3660 ctccaagaca accaggtgaa gtccaagagt tagtaaaata aggattgtgg ctcggctgtg 3720 gcctaatgca aacttgcaca accccaggaa accgaaaaaa actggaagaa gaattgcaga 3780 atggggtgcc aggttgaaag accttaacct ttgcactcaa attcctccca cacccagaag 3840 tccaggtccc gaccgcacga cgccgtgctc actgctgggt gctgcgcctg agtccgcctc 3900 ctgcggcttc ccggacttga ccccgcccac gccctggtcc cgcctcctgc cgacgccggc 3960 acagaccccg gtgacggaag tgacgtaagg ccggggctgg agggcagtgc tgggctggtc 4020 ccgcaggcgc tcggggttgg agccagcgac cgtcggtagc agcatggctc tcctctttct 4080 cctacccctt gtcatgcagg gtgtgagcag ggctgagatg ggcaccgcgg atctggggcc 4140 gtcctcaggt accgtcgttc gcggcagggc tgcggccggg tcgggacgag agggagggag 4200 ggatccgccc cagccgggaa gccccgcccc gcttctccga ggtcgcccta gcccgggacc 4260 cctgcgtcgg gccctgagcg ggcgacgggg acgcgaggtg gggtcgcgag actggggctc 4320 cgtggtttga actcgtcggt tgcctttctc ctgcctcttc cctggttgcc gccacaacaa 4380 atcacgccgc ttgtttttcc gactcttgta atccactttt aaatacgatt atgcgctgca 4440 gaaggacgtt gctggtcaat gtgggacagc gtggtctcaa agattataat atcgtcttct 4500 tactgtacct ttttttatgg ttagatacgc agatactcac tagtatgcta cggttgcctg 4560 cagtgttcag tgcagtgaca ggctgtacag gttcatgacc taagagcagt aggctgtgtc 4620 atctgggttt gtctgggttt ctactgcccg aagaaattgc ctaaggggac atttctccat 4680 ttctcagaac ggatctccat cgttaagtga cgcatgactg tgagtgtcct gatcagcatg 4740 tggcatcgtg gtctcagttc gtggcttaaa tacttctttc ttagcggggg tggggggggg 4800 gactcaaagt gcttagggta ggaaagtatg cattatgtcg ccagctgaga agtaagtggc 4860 agctgagaag taagtggcag tccgtcttag gtctcagtct ggaagtggtg gattgccggc 4920 actcactagc agatttggca gtaggtctct tcgtacgggt ttgttcttcg gagcatttta 4980 tttccacctg cttattgaca tttcctagaa aacaaatgtt gagggtggct tttctgaagt 5040 gcagagtaat tgtgattacg agctccagag aaaacagtaa accttttcaa ttcagttttc 5100 ttttttttat tatgatttat gattcatagg gactttgaaa ggtccacgtt ttggttctgt 5160 gttcacaaca atcttctgga gtaggcgtta tttgtctctc tctttatgga cgagaaattg 5220 agttacggag aggcctgccc aactggcaga gaggtcatag ccactcagtg gggagctgct 5280 caatttggct ccagagtcca agttcttaac tgctcttcta ggctctagtc agtcatccac 5340 atgggcagtt gcagtacttt gcggtaagag cttgcactta ggcacagact agtggaaagc 5400 aaggcggggt gcttgtctct tttagggaag tcagtgaaga cttcacaaag gtgaccccag 5460 tgttggaacc agaggtgcag agactgaggt gcagaggtgt gatagcatgt tgaattcgtg 5520 gaatattaag cgttctattg taaagtgtgt gtgagagctt acaaggggag gagataggtt 5580 aggtggagaa gtaggaacca aataatgaat tgccatatta tgtgtaccag caagaaacct 5640 agctttatta acttaagcaa aaagaggaat ttattgtctc agaaaaacca gagttaaaca 5700 gaaaattata gatgccagct gggtctcaag aggctagaac caggaaccca aactgctagg 5760 actctccatt tcttatttct gtctgggtat tgacagcttt ccacacttgg caggaatcat 5820 ggatgctgac agcttctgag ttttgacatc ttgcagcctc ctccattcta acaaaatgcc 5880 agggaaaggc tctgatttgc ccagctaggt tcaggtgcca gcttgcatgg gaaggacatt 5940 ctcgtagagg gttgggttgg gttaccagaa gaagcggaat ggcgtgcctg aagtacaggt 6000 gttgagccaa caaaataata cacccactat gccttgctga agtcagtcat tatttactaa 6060 tgctgacatc attggattat caattacaaa gggaaatgat tttatgcagg atcagacatg 6120 ccatttagaa agaccagtca tcttataaaa tagtttgagg gtaaacacaa ggcccattag 6180 gaggctgctg cttaagttgg agtcgataag tagtttaagt ggagaagagg acagttagca 6240 gaacatttag aaggaagagt tagtaggcct ctgactggat gtcagcagag aaagaggata 6300 acgctagcgc aggcatagcg ttaaagcaag tgccaggccc ttccttctga atacggggcc 6360 ctatgagtgc accgtgtgac ccctgtgagt gcacaatttg cacgtccttg tagcttgctc 6420 tgggaggagg attgcctgtc tgcctcccgt gggaagatca actgcagcct tctatattct 6480 gcagaaactt ttgaatttgt ctctgtacct ctcgttctaa aactggggtc acttttgtga 6540 agcctttcct gactccccta gaagagacaa gcaccccggc cttgcttttc attagtttca 6600 gtgagccctc tttttaaagt gcatttctaa cacatttctc tagatttttc agtgcatttc 6660 tctagatttt gttgtgttcc ctgttctttt aaagttaatt taaaacacaa aatctccaaa 6720 gtaggaagaa accaacaatg ctctggctct tgcttagctt cccagcctta ttcttactgt 6780 tgctgggctt gactctatag ttgaagttca tcttagatga gaggacctgg taagaacagg 6840 gaagcagaca agtgaatatg gtgatatagt cagtctccag gaaatccagg tgtttaggaa 6900 tgtattgtgt tacattcatg ggctttcatt aatacttctt aagtgaaaat aaaatctgtg 6960 tgtgccactt aatcagtagt atttcactct tcatgcactt ttacacataa aagtagtata 7020 tatttttaag tgaatcaggg tgtgccactg gctttcttca tccagatgag tgtcccactc 7080 tgagtaatag ctgaagagct aggctggact tcacaccaac tggaaggctc ttcactgacc 7140 ttgtacagtt tatcagctat gcttcctact aattatctga ctttaggata aagctctgag 7200 tactgtttaa atttttcact atttctttgg attctatagt tatttcagag attcaaaata 7260 cacaggataa tactttttaa ttatatttat tgaatagggt attaaatttt ctttttcatt 7320 ttattctgat tcataattca cacagacttt gaaaggacca tattttggtt ctatctaaaa 7380 gaaaacctag gctgggggca gtggctcatg cctgtaatcc cagcacatcg gcggaaggat 7440 cactggagcc cagaagttca agaccagcct gggcaacatg gcaaaaccca tctctacaaa 7500 aaaatacaaa aattaaccct gtgtggtggc atgtgcctgt agtcctagct actggggagg 7560 ctgaggtagg aggatcactt gagcccagga ggtctaggct gcagtgagcc atgattgtac 7620 cactgcactc cagcttgggc aacagagcaa gactctgtct ctcgtgtttg tgtgtgtgtt 7680 tgtgtgtgtg tgtgtgtttg tgtatgaaaa cctaaagaaa agtaataaac agctatctta 7740 caaaatttgg cagcatacca gtagtgaagt tgctttacaa accagatgag taattaatgg 7800 ttcaattttg attgaacaaa gaatcagaat gaggtttgaa ccagaatgaa gtgaattcct 7860 aacagaaagg aatttacttg taattttctt attaaagggg tgcctttatt ttagattatc 7920 ctaatgcata gtaatcagaa tactaggtat ctgtttaggt cttgtgtatt tttaagaaat 7980 tattcatttt aatgcattgt tcaacttttg cagtggccat aatataattg tgataatgta 8040 ataaaatcag gaaacattac ctgaagcaga tgcttttgaa gtcaccagaa aatagtcttc 8100 tccaattcca atttaaatat ggaggtaatt taccccttat tttattaaac gatcacaaag 8160 atgttggaag catttttctt aatgaaaacc aacaaaactt tgcatattgt caagttacgt 8220 aacttctcta agcctttgtt ttctcattgg taaaatatgt atggtaacat tgtctccctc 8280 acgggatgga tgtgatgagt atctgaggta atgcattaaa aaggtaagca cactgtgtgt 8340 cttgtagtaa atacttaata tgttagggat tattcaataa tgtgcattat tattttaaca 8400 aatatatctt taaatattaa attcccttag tcatatgtga gaaattgtgt tggcttttaa 8460 aaaaacaaac ttaaccagat cagtatgaga aacttaaaac aggctcagca gctaactcat 8520 tcttccatct cttccaacag acaaaaaagt ttggtttttt agtagactgg aaaggcctgc 8580 attgtttttt catttcctca actgtttttc aattcagaat gagttagtcc aaaagagaat 8640 tagtgttgag aaatgacttc tctcagggac tagattggtc tggtccagtc agaactaatg 8700 ccagaccaaa cccaactaga tgaaattgga taaataggag taagaataaa gccctggatt 8760 tcaagtcaga aaagtggctg agcagatatt tattggataa gccccaactt ggtggtggtg 8820 cgagggaaaa atatagggga ggttagttac ttacaagttc actgcaggcc actatgtgtt 8880 atgcctggct ctttaaaaag aaaaatttca attagaagat ttcaatgaac acttgaaaca 8940 tcacctgcaa gaattatcaa aatgtctgta ctcagcttaa acctggggga tgcggctcct 9000 tgctacggtg aaagttggaa agcattaaga aaaatagtca ttttatcacc actggaagcc 9060 attcgggagg gagattgtaa tcaaatatgt tctgacaaaa atacaaaggc cttgatttgg 9120 tacttttcat ttattcagtg tggatttctt atgctggttc tgtttgctgt gcagatgaaa 9180 cctttgtgac agtccccatt cctgtccctg tatctttaac atggggaagg tgttagatct 9240 tctctgagag ttgttcttaa ggaaaagatc cagatgcttt aatttgaaac atttgttttt 9300 gagttgtagt gtcagatgac ttagaatcca gaacaacagt ttacttcatc ctcacttcct 9360 tatgcttctc agcgtgctct tcccatgaag aggttggtga gcaggttgtc agatgatttc 9420 tgcagagtcc atagccctgt tccaccatag atgtacaaac cttttataca gtctttttac 9480 actagtgtgt taagatgggt gatgaactcc tcgggctgtc cttttttaaa gagacacaga 9540 tgcttttggt ggttacagga tggactcagg tagcagccct ggttcctgtc acccagctat 9600 gcagaggaaa tggccagtgt cttcagacta tactctgcag gtatctgtag tttgccatcc 9660 accctacatt caccaacatt tattgcagag ctatcacgac tgctattttt tggtcctttg 9720 tgacattgtt atggtcccgt cgatagcact gctctgtatc acctgctgat cttcactggt 9780 cttttgtgtt gtctctcttt tttcatatcc tgtctcttct ggcttatcag ctttctgagg 9840 aaacagttgg ctttaaccag ggccctcctt tttttttctg cattgtaatt aaagtgttag 9900 gaaaaacaaa ttcttctctc ctgtgtttag ttttcttaaa ttatcagctg tgagttgtag 9960 gggagttctg ggaattagat cttaaaatag ctaaagttta ctccggatag tatgcagatg 10020 taatacacta aataattctg agtccatttc caaaagcaac atgagttaca caaaactatc 10080 ttggcatttt caagagatgg gaaaatctgt aagattatct tatttcatga aatcactctg 10140 gtaagatctt ttcttaagcc ccagagataa taatactctt ttgctgtttc tacatatatt 10200 tttatttaca tagtgtaaat gtattagaag ttaattgtaa gccttaatta atttaatctg 10260 aatatataac aatttaaacc tcacattaca aaattctgaa gcttaagaaa actgcttatg 10320 tctgtcatat tattttggca aataatattt ggcaaaaaat aatatttggc aaaacaaagg 10380 cagatgtcta ataaccacac agtttctcct ctggtagcat taaaaaccac agattacatc 10440 ttttggtgta aatttataag tttttctttt aacctagtag taatttcttt ttgctataaa 10500 ccattgaaat attttcctcc taccacctgt ctgtcttgct ttgaaagcta tgatttaagt 10560 ccttgtccta gtctaaccat atcaggcttt tacttaaatt ataggaaaca tactttgatt 10620 cacgttaaaa actaatctaa tgctgcacaa gcctactcct gtttgtagtt ttccattgtg 10680 tgattcagta gccccttcct aagtacaaat gcactttgtt tgatattcta gtatgcagat 10740 tgacttctat atgccttttt ctcacaggaa tttcaggttg agcgtatacc attataaagc 10800 cattcctact gggtataatt aaaaagagag ccttaggctg ggcccggtgg ctcacacctg 10860 taatctcagc actttgggag gcccaggtgg gcgattcatg aggttgggag tttgagacca 10920 gcctgaccaa catggtgaaa ccccatctct actaaaaata caaaaattag ccatgtctgg 10980 tggtgcaaac ctgtaatctc agctactcgg gaggctgagg caggagaatc gtttgaaccc 11040 aggaggcaga ggttgcagtg agcagagatc atgccattgc actccaacct gggcaacaga 11100 gcaagactct gtctcaaaag gaggggagtg gagcttaaaa ggaagcaaag gatttccagt 11160 cttggaaacc tagaggaagg ggggtccttg ctatgtagtg gcagaaagct gagtgacact 11220 gtagccttca gtgacaccaa aagtagagaa tgtctgagga gctgtgtgat ctagctcagg 11280 aggtttctaa ccagagtgct gaaagtgcca cctggtttct tcttgcgtct tatagtaaca 11340 ggtgagactt tgctcaatca tgggaagaac tgttaaaaag aaaccaggac cacctggttt 11400 gaaaattctc agcctctcga gaggcaaaaa aatgccaaaa tgaagaaatg gtctccgaag 11460 gaaggctcag atttagtatg agttttctta gcatcaagta cctctgtttt ccggttgaaa 11520 aagtatattt atatgtgtga ttgttatttg atattaatgt tgattgaacc caggtgaaga 11580 ggaatgactt ttatttctac ctaaaatatg gattgtgaag tatttgagtt gtttcaggag 11640 gttgagatgc aaacgtgtga tagaaaaaag accctcttaa ggaagcctca gtatcccctg 11700 ctatgaatgg ccatgggaac tctacaaaaa acaaagagcc aaggctactc tggcaagaac 11760 agtgccttag ggatcaccta caaggaagtt gcttagagaa ccagagatgg caggtaccaa 11820 gagccttcag ctgcgcctct ggaagcagaa ggagaaaacc taggtgtctg gtcactggcc 11880 acaaaaaggt caagttagta gtatgaagta tttgaatctt cagaccctcc ccagcttgat 11940 aatctcaacc ttttggccca aattattaca ccattttggt aaataatata gaagttagat 12000 cttttggcaa taaccaaatc tattcatttg tttgagggga gccatgaaat atacaggttt 12060 actgtaaagc attctgtttt taagttagtt tttcctcaac ttttttttta aagaacactt 12120 aaattgtaaa cataaagcag ggtttctcaa ccttggcact gctggcattt ggggctatat 12180 tattttttgt tgtggaggct gtctgatgca ttgtaggata tttagcagca cccttggtct 12240 ctgccctcta gatgccagca gtacctcccc cacacacttc ttctccaggt tgtgagagcc 12300 aaaaatgtct acagacattg attgcctaat gtctcctggc aacaaaatca cttcccacag 12360 agaaccactg caataaagga aaaaataatc agaaaaggaa aataataatg gtgatagcat 12420 ttaattgtcc attagtgcct attatttacc taaaaaactc ttaatccatg ttctgctttg 12480 aaaatatatg tgttaaataa ttgtaaatta aggcaatata ccagtttggt cttgaatatt 12540 ggaatttttg tttggggaaa atatagattt tcccttcagc agtaagcaaa tatcttaggg 12600 atatgaaagt aacacctcat gacagatcag gaaatctgaa gtatttagca gtgtggccaa 12660 gaaagaaccc aaatttatta atgtttgacc tagggagtat ttttcttatt taagcagtct 12720 gcttgatata aatataaatc atcatcatta tcatccaagg cactttggaa aaatagaaac 12780 ttctgtccct ctcacaattt ttgaggatga ttataagtta tgtttggaag ccactgttcc 12840 tatccacatg gacattcatt tatttgtttt agttatataa gtaaatcatg tttaatagag 12900 tgttccaggg aggtgagatt gaataaccat aaatttgggg tcacatcgct aactgtgtac 12960 attcatgttt atctgtaaag aagtgaaggg agaaaggaga agtgtgtgtg ttcagttttc 13020 caaggggtgt tggataacaa ccttttacac caaacagtgg ggaaaaatga ctcctttttt 13080 tttttttcta agagaactga atgcagtggc acagtcacag ctcactgcag ccttgacctc 13140 ctgggctcaa gcaatcttcc tcagcctccc gagtagctga aactacaggc acaggcctct 13200 tcacctggct aattttttaa ttatttgcgg agacagggtc tccaactcct gagctcaaca 13260 gatcctccca cctcagcctc ccaaaatgct gggattacag gtgtgggcca ccgcacctgg 13320 cccatgacta cttttaataa ctagattttg gtagtgtctg attagatgtt aaaatgactt 13380 ctgtctgttc tcttacgtga ctcaagtctt atcaacagat gttttctgaa agagtgaagg 13440 caagaagaaa tgttgggtat gttttgtaaa aagccctgca caccccagag agcagagtgt 13500 tgatgaaact ggaatgtgtg gctccattgt tcaatcttta ggtatctatt cctgtactat 13560 agaaaaagtg tggaaggcat aaatagtagt atgtgtagag ttgagaggtt gatggtattt 13620 tagagtgaaa ttggccatcc tcattgtagc cagttcctct gtagacattt gtattagtct 13680 gttttcgtgc tgctgataaa gacataccca aggctgggaa gaaaaagatg tttgattgga 13740 cttacagttc cacatggctg gggaggcctc agaatcacag caagggcgaa aggcacttct 13800 tacatggtgg cggcaagaga aaaatgagga agaagcaaaa gcagaaaccc ctgataaacc 13860 catcagatct cgtgagactt actcactatc acgagactag catgggaaag accagccccc 13920 gtgattcaat tacttccccc aggtccctct cataacacgt gggaattcca agagatacaa 13980 ttcaagttga gatttcggtg gggacacaca accaaaccat attatgctac ccctggcccc 14040 tccaaatctc atgtccttac atttcaaaac caatcatgcc ttccaaacag tctcccaaag 14100 tcttaactca tttcagcatt aacccaaaag tccacattcc aaagtcccat ctgagacaag 14160 gcaagtcctt tctgcctgtg agcctgtaaa atcaaaagca agctagttac ttcctagata 14220 cagtgtgggt acaggtattg ggtaaataca gccattccaa atgggagaaa ttggccaaaa 14280 caaaggagtt atagagccca tgcaagtcca taatccagtg gggcagtcaa atcttaaagc 14340 tccgaaatga tctcctttga ctccaggtct cacatccagg tcatgcggat gtaagaggtg 14400 gcttcccatg gtcttgagca gctctgcccc tgtggctttg cagggtacag gctccctctc 14460 agctgctttc acaggctgcc atcaaatgtc tgcagctttt ccagaagttg tcagcagatc 14520 taccattctg gagtctggag gatggtggcc ctcttctcac agctctacca ggcagtgccc 14580 cagtagggac ttgtgtgagg gctccgacct cacattttcc ttccacactg gcctagcaga 14640 ggttctccat gagggccctg cccctgcagc aaacttttgc ctgggcatcc aggcatttcc 14700 atacatcttc tgaaatctaa gcagaggttc ccaaatctca attcttgact tctgcacacc 14760 cataggctca acaccatgtg gaagctgcca aggcttgtga cttccaccct ccgaagccac 14820 agcctgagct atatgttggc ctctttcagt cacagctgga gcagctggga cgcaaggcac 14880 caagtcccta ggctgcacac agcacgggga ccctgggcct ggccacaaaa ccactttttc 14940 ctcctaggcc tccgggcctg tgatgggaag cgctggcgtg aatgtctctg acatggcctg 15000 gagacatttt ctcgatggcc ttggggatta acattaggct tcttgctact tatgcacatt 15060 tctgcagcca gcttgaattt ctccccagaa aatgggtttt tcttttctgt ctcatagtca 15120 ggctgcaaat tttccaaact tttatgctct gcttccctta taaaactgaa tgcctttaac 15180 agcacgcaag tcacttcttg aatgctatgc cacttagtaa tttcttccac cagataccct 15240 aaatcatctc tctcaagttc agagttccac aaatctctgg ggcaggggca aaatgccgcc 15300 agtctctttg ctaaaacata gcaagagtca cctttgctcc agttcccagc aagttcctca 15360 cctccactga gaccacctca gcctggattt tattgtccat attgctatca gcattttggg 15420 caaagccatt caacaagtct cttaagtaaa gttccaaaca ttttcctatt tcttctgagc 15480 cctccaaact gttccagtct ctgcctgtta ccctgttcca aagttgcttc cacattttct 15540 tgtatctttt cagcaatgcc ccactctgct ggtaccaatt tactgtatta gtcctttttc 15600 atgctgctga taaagacatg cccaagactg ggaagaaaaa gatgtttaat tgaacttaca 15660 gttccacatg gttggagagg cctcagaatc atggcggggg agaaaggcac ttcttacatg 15720 gtggcagcaa gagaaaatga ggacgaagca aaaagcagaa gccccaataa acccatcata 15780 tctcatgaga cttattcact atcacgaaaa tagcacagga aagactggcc cccatgattc 15840 aattacctct ccctgggtcc aagccacaac acatgggaat tctgggagat acagttcaag 15900 ttgagatttg ggtggggaca cagccaaacc ctatcaacat tgaatacaat ctgtagtatc 15960 aaatattgaa aattctagtg atgaaatgag gattattaat tcaataatca gtgcatttga 16020 ttataaaact tctatcatat taagggaatt tattgcttca tgctgtattt gtgtttcctt 16080 tatattgcaa tgatgtttcc ataatttaaa acgaatcaga actcttctat agatttttca 16140 ataaaattaa tttctagatg tctactcttc atttaaataa ttttggtatc ctggtgaatt 16200 ctacttttct tcaaatatac atatctgggc aatgtggcat cttacaataa ggctttccaa 16260 tattaatgtt agtttcttac aattgtccct tttacttttt tattttctta cagtgcctac 16320 accaactaat gttacaattg aatcctataa catgaaccct atcgtatatt gggagtacca 16380 gatcatgcca caggtccctg tttttaccgt agaggtaaag aactatgggt gagtgtcact 16440 cttttattta tcctttttat tccatttttg tttaggtcct tggaaattcc acaactgtgt 16500 tctttcatca gccttcccac gtggcaaaac attctaaact gcttatagag gtccaaaagt 16560 actaaagatg caaattcttt gccaaatatt tcttgcctct tatttcctct tccttatcag 16620 tattgaaata gagaagtatc actaaacttc tgagattagc atgacaaata aagctaatag 16680 ttactatgtc atcttcctgt agtgtatttt tagtagaaca atataaattt caaaaaactt 16740 ttttgacaac tgttagtttt tttaatgtgc ttacttctat aattaacata tataaatgga 16800 atttagagtt gtttaaaaaa acagattttt aaaaaattga atgtggaatg tggcttgcat 16860 aggtcttaca ttttgaattg agcctcttta ctgtaacaga gtttgtagtt cttctaacta 16920 aagagtaagg gtttcctact ttctctggcg tctccatcct attcttagct ctgctctttc 16980 taccgctttg tgctgtgaat aaaaagcaaa gcacagacag aaatggtttg agtttattta 17040 ataacaataa aagttatctt cgcatttttt tattcttttt agtgttaaga attcagaatg 17100 gattgatgcc tgcatcaata tttctcatca ttattgtaat atttctgatc atgttggtga 17160 tccatcaaat tctctttggg tcagagttaa agccagggtt ggacaaaaag aatctgccta 17220 tgcaaagtca gaagaatttg ctgtatgccg agatggtgag tagaatgtgt acacatgtaa 17280 attttaaatt ggaaaatatt tatacatgtc ttctgtatgt ttgcttttat tagcagttgc 17340 tgaaaattat cacaatgctt aagaaacact tggaggaatt tagagtcagt acaggttgag 17400 catcccaagt ctagaaaccc agaatgctcc aaaatctgaa actttttgag cgccagcatg 17460 gcactcaaag gaaatactca ttggagcatt ttggatgttc agatttagaa tgttcaactg 17520 gtaaatataa tgtaaatatt ccagaatcca gaaaaaaaaa ctcagaaacc gaaaatacta 17580 ctgatctcaa gcgttttgga tatgggatac tcaacctgta tcctcccctt ctttcaagcc 17640 ccacaaaata gggctaacag gtggccatca cttagctgct tctatgccat gacatttcca 17700 cttcatacaa ttatgggtct gtgctgccca gcggtcccct gcaccaccct tgcccttccc 17760 atcttttgtc attcatttgt aagttatatt cactaaggac tttggcagct aactcttatt 17820 ctgaatgcct ggggaacctg aataccacct taatacatta aaaaattgca gagttcctta 17880 ttctcttcag ccactgtgat tttcaggtct tcagtcttcc agcatcccac tcccatagtg 17940 tacctgatgt ttgctgtcac ctcaaactct tgaatcttta attccagttc ctgtattcca 18000 actatgacat cctgccttag ctctcagacc ctgactcctg ggtaaaagac acctttagtc 18060 acttgactcc tccctagtct tctagccctt gggtcccttt ccctctagcc tcacttcctt 18120 tctctgctcc tgccttttag attgtgccac ctcggtctct tctacaggct cttcctctcc 18180 ttacccttta aatacttttc ttccccagag tccacaattt tgtcatatga ttaccacacc 18240 atctgcatgt tagatttttt tccatataga tttatcaagg gcctactacg tgccggaaag 18300 tatactgggc cctgaggata cactgatggg ttaaaaacag acctgcctgg cacacagtgg 18360 tacacagtaa tatgtgaaga ctagataaat ggttcacaac aaagtctcaa acatcaaaga 18420 cctaataaat atttatggaa taagttctga tttttcacag taaaaagagt tttgatgagg 18480 gaagaaaact aatgccattt ctgaaacgtt ttttagaatc caggcttctt aatccacaga 18540 agcaaagata aaggaaaaat tacttaatag agtctatatt atgtgaagtc tttttttaac 18600 caaagtttat aactattttt atttacaact agaaatgaaa ttaaactgct aaataggcat 18660 tcagattaaa taagagtact tcttgcctga gatttatgaa acatttgttt attaaggacc 18720 ccgagactat cttctagata tttgtggagt ggtttaaatg tggtcctgct ttagaacaac 18780 cagaacaaaa acaataggtt gattgataga ttctggtaat tttatgaaat gtaccattta 18840 gttccaaggc cagtatttat tatacttcct cctcctcctt ccccaggaaa aattggacca 18900 cctaaactgg atatcagaaa ggaggagaag caaatcatga ttgacatatt tcacccttca 18960 gtttttgtaa atggagacga gcaggaagtc gattatgatc ccgaaactac ctgttacatt 19020 agggtgtaca atgtgtatgt gagaatgaac ggaagtgagg tatgtgtttc acatttttca 19080 taatggaaat tcttgtgtag ctagcaaaag ttgttccttt ctgtagtgta atgaaaatag 19140 gatgcttata aatattcaag caagactcac agatcataga tttgataaga aaaatatgtg 19200 aatgctatta aagcaaaaat gatacagagt tagtcactaa cactgaccat gtgaatacat 19260 ttagttttta tttgcttcat ttagcagaat ggctctaatc taggattttt cccagtacac 19320 ctccattgct ccactggtaa aatgaggatt ttcttcctca ttggcggtga cgtaaagtaa 19380 tattaatact tctgtttccc accagctaaa tttctggaca aatgaatagg acaaatgaac 19440 atttgttagt ttaacaacta acaataaaca ttcctgcaat ttgagttttt aaatttacca 19500 tttttataag gaatataaca gatgcatagt atcgtgctgt gttgaagtaa caaattgact 19560 tattgatagt aaagctattt ttagacatgt tcaagtcaat catatatatt tcttcagttg 19620 tttgaccagg actaatatgg tgattttttt tttttttcag attaaaagaa gctgtgcatt 19680 ttcactgttt tcttttttca tctagatcca gtataaaata ctcacgcaga aggaagatga 19740 ttgtgacgag attcagtgcc agttagcgat tccagtatcc tcactgaatt ctcagtactg 19800 tgtttcagca gaaggagtct tacatgtgtg gggtgttaca actgaaaagt caaaagaagt 19860 ttgtattacc attttcaata gcagtataaa aggtaagttc ttgccatttt ttttctaaat 19920 atagagggag cagtaactaa aataggatca tgtgaggaaa gcaaactcat ttgcagtttc 19980 aaaagatctg ttttaacaat attcatttgc attagttcca ttccttagag aagttcaaaa 20040 ttaaaaataa agcagtttct cacacattta aagatataga acaactaatt tggaaagcca 20100 tgtgatggcc ccttgaccca ggaaagaaaa aatatataat aagtacataa aactacacaa 20160 atgtttgcat ttcaggatta tgaattataa gtcagtttgt tacttcacca tagcaagatg 20220 ttgtgaatcc actaaaatgt tctttatggg gattgtaatt taaagactga agagcaggaa 20280 cgatcactgg ttacaactgt atgacgtgga ctggacaaag actcgagatt aatatccaaa 20340 aaagtttata gatgggtttg aataatagat aatgggtcat ttttataaca ttttacaata 20400 attaatttta tttcatcttt taatgtgggg ggtggggagg tagagatata tatgctagaa 20460 cttgaacttt agaactgtta ctctcaggtg tcaggaacct taaagaagtt tggacttttc 20520 tttgcagtta ggaaatgtaa gtgttcatta tagataattt ataaaatatg aaaaaccaga 20580 agaaaacaaa ataatctgta gttctaccat gcaaaaatac cccattattc acagtttgat 20640 attttcttta gtttttctac atagacatga tcatattgta tagtatatat gattttgtgt 20700 attgctttaa gcatatattg taactcatgc tttgatgatt atctttgtgt gtgtgtgtgt 20760 gtgtgtgtgt gtgtgtgtgt gtgtgtgtat acatatattt ttttccttag gattttttta 20820 gaatagattt cataggttta aaattttatg atgcaagtat attttcgagg aatggaaata 20880 taggctgaag agccagaaca aacacctaaa attcaatact aataacttca agccattata 20940 tgaaggtttc aaataggtga tttaaagcag tgcaatgcat tttatgctaa ttttcgttat 21000 gctatttgaa aaataaaacc cttcttaatg ctcaaaattt gaaataatgc aaatgccgca 21060 gatttgtttt tagcctgaat tatacactgc actgatagta tggccacacg gtggcattgc 21120 cgtggctcct gagcagcagc cctgcctgct tgcctcaatg ccttgcagcc agcacttgct 21180 cagcccgtgg gacttctaac agcactaatg ttggttttgt agttattttc ctttttactt 21240 ttgtaatgat ttaacttttt tcacaccctg taataagcgt aatagaagca ttcattagta 21300 acttccatag ttagaaaatt ctgggtaaca aaatgcccta aaggatgccg gacattgtca 21360 cagtatgtta gagtgctgtt aatttggaag agagaactat gaaaaaaaaa aaatactgaa 21420 gattgtgctg caaaaataaa aggcagcatt acagcaaagt acacaatggt gattctagat 21480 gtatgtgctt ggaaacaaat ctttcctatt ttatattatt tatgtctggt ttataactaa 21540 ggggacttta ttcctgtgca gcattgctgt gtgcatgtac atctgtgaag ctttataagt 21600 atttgcagtc gtgaaataaa tgtaacagga aattctgagg agtggggcta aatcatctta 21660 agagcaataa acattgccag aatttctttc tttttttttt tgttgttttg ttttgttttg 21720 aaatggagtc tcgctctgtc accaggctgg agtccagtgg cacaatctca gctcactgct 21780 acctccactt cccaggtcca agcgattctc ctgcctcagc ctcccacgta gctgggacta 21840 caggcgcatg ccaccacacc cagctaattt ttgtattttt agtagagatg gggtttcacc 21900 atgttggcca agattgtctc gatctcttga cctcgtgatc cgcccacctc gacttcccaa 21960 agtgctggga ttacaggctt gagccactgt gcccagctgc cagaatttgt ttctaaggag 22020 aggtattttt aaaattattc ttttgcattt ttaaccgaaa gaaatgcata accctggaaa 22080 cacactgtat gtagggctgt aaggaaatta ttgtagaaaa gctattttaa ctatgttgtg 22140 tcatagtaga atagtcccag aaagttctag aattgcagag ctgggaagaa ccatattgct 22200 atctaagaca gatacctaat tttttactca gatttcctaa cttctgcttc cttcagtgtt 22260 ctttaaaact ctggccctat tcactaattt gtaaatctat tcaagaatgg cactaagact 22320 ttttgataga taatagcagt attccatctt aattgtaact tgtgatttct gcctttttta 22380 aggttctctt tggattccag ttgttgctgc tttactactc tttctagtgc ttagcctggt 22440 attcatctgt ttttatatta agaaaattaa tccattgaag gaaaaaagca taatattacc 22500 caagtccttg gtaatgtatt taattttttt atatacacta aaaagttaac ttgaaccttt 22560 tttgatagtt tcttaaatta tgtttttaaa gtagaatgat attcaacaag atgtcacctg 22620 ccatcaatca gtcagtctac cacacatgca aaatcacata ctgtgtcttt tgctgtgcag 22680 caagtatttt gttttttttt ttattctcag ggaaaaacac ggtgagacaa ttctgttaaa 22740 gatcttaaag tctaggggga agaaaagata aatatacatt ggctatataa cgtgtgatag 22800 attcaaagat ttatacctga gcccatgagg actttgtcca gctttggggt ggggttagtc 22860 atggcttttc cagaggcaat gcctgagcag tcttgaggga catacaggag tttaggcact 22920 aggggacaga atgtgctgag gtccagagcc aagaaagcat gactttgaaa gctctgcaaa 22980 tacagaaaca cttcattatg gttggagtat gaaaggaaag ttggccaaaa atgagactaa 23040 cgtaggagtt tgacgatttt aatgtccaac atttcaaagc ctttgatgta aagaacattt 23100 aaaggtaaca gaatagtatc tatttgagtc aaatatgaca ggaataatat ctgtgatgct 23160 caaagggcac attaaaattt gctataaact acctttaaag tggataagaa ctctcaacag 23220 ttagtagtca actcacaaat gaattataat attttattct tactagtaat taagcacaaa 23280 gtaaccttgc agtatgccat tttacacgcg ctagagaaga aaaaagaaac ctggatatcc 23340 agtagtacca gcagtagcat aacgggtaaa tgctggtgaa aagtaatcta gccctggtaa 23400 gaataaaagc acacagtggg tattcagtgt aatagtgacg taaacatacc atttctttga 23460 tatggtcact ccactcttaa aaaatgtatt ataaggaaat aatttcataa ataaaaagat 23520 gtacaggata tttaacatgt tccatagcta gtaaaaattg gaaacatttc cccatctatt 23580 aaattattta tcagtataaa gaaataatat gagctactaa aaattataat gatgaaggca 23640 agtaatgaga atattactta agtagatgtg cccactttgt aaacataatg tgcgtacatg 23700 tggaagataa ttgtattagg attctctaga gggacagaac taatggaata tatatataaa 23760 gatatatata taataccaaa taaactcccc tttatataaa tctatatttt atatattata 23820 tattatatat attttatatg ttatataata tatattatat attatatata atatattata 23880 aattatatat tttatataat atattataaa ttatatattt tatataatat aagttatata 23940 ttttatatat tatattataa attatatgtt tatataatat attataaatt atatatttta 24000 tgtaatataa attatatttt atataatata ttataaatta taaattatat attatatatt 24060 ataaattata tattatataa tatatattaa ttatattttt catatatata tatataaagg 24120 ggagtttatt tagtattatt tagtattaac tcacacgatc agaggtccca caataggcca 24180 tctgcaggct gaggagcaag gagagccagt ccaagctcca aacttgaaga attcagagtc 24240 cgatgttcaa gggcaggaag cattcagcat gggagaaaga tgtaggctgg gaggctaggc 24300 cagtctctct tttcacattt ttctgcctgc ttacattcta gccatgctgg cagctgatta 24360 gattgtgccc attcgggtta agggcgggtc ttcctttccc agcccactga ctcaaatgtt 24420 aatctccttt ggcagcaccc tcacagacac acccaggatc aatactttgt atccttcagt 24480 ccaatcaagt tgacactcag tattaaccat cacagtaacg tacaaaaagc aacatatatt 24540 agtaagatat ctgatggctt tttaaaaatt ctaaaacttt gtttttaata ttactatggg 24600 acctttcatt aaaaagaaat ggcaacatct gattcaccca ttatcctaaa tgtgccattt 24660 ggtggtccat tacttcagac ctttgttttt tttgagggta ggcacttaag cttaacaatt 24720 ttttatcttt aatcaatttt tctccccata gatctctgtg gtaagaagtg ctactttaga 24780 gacaaaacct gaatcaaaat atgtatcact catcacgtca taccagccat tttccttaga 24840 aaaggaggtg gtctgtgaag agccgttgtc tccagcaaca gttccaggca tgcataccga 24900 agacaatcca ggaaaagtgg aacatacaga agaactttct agtataacag aagtggtgac 24960 tactgaagaa aatattcctg acgtggtccc gggcagccat ctgactccaa tagagagaga 25020 gagttcttca cctttaagta gtaaccagtc tgaacctggc agcatcgctt taaactcgta 25080 tcactccaga aattgttctg agagtgatca ctccagaaat ggttttgata ctgattccag 25140 ctgtctggaa tcacatagct ccttatctga ctcagaattt cccccaaata ataaaggtga 25200 aataaaaaca gaaggacaag agctcataac cgtaataaaa gcccccacct cctttggtta 25260 tgataaacca catgtgctag tggatctact tgtggatgat agcggtaaag agtccttgat 25320 tggttataga ccaacagaag attccaaaga attttcatga gatcagctaa gttgcaccaa 25380 ctttgaagtc tgattttcct ggacagtttt ctgctttaat ttcatgaaaa gattatgatc 25440 tcagaaattg tatcttagtt ggtatcaacc aaatggagtg acttagtgta catgaaagcg 25500 taaagaggat gtgtggcatt ttcacttttg gcttgtaaag tacagacttt tttttttttt 25560 taaacaaaaa aagcattgta acttatgaac ctttacatcc agataggtta ccagtaacgg 25620 aacagtatcc agtactcctg gttcctaggt gagcaggtga tgccccaggg acctttgtag 25680 ccacttcact ttttttcttt tctctgcctt ggtatagcat atgtttttgt aagtttatgc 25740 atacagtaat tttaagtaat ttcagaagaa attctgcaag cttttcaaaa ttggacttaa 25800 aatctaattc aaactaatag aattaatgga atatgtaaat agaaacgtgt atatttttta 25860 tgaaacatta cagttagaga tttttaaata aagaatttta aaactcgttt ttgtattcat 25920 ttattcaagc acttaggcag tctttcatag ttaagcagaa tatttttata tccactgctt 25980 gtttccatgc attaatgcta 26000 11 20 DNA Artificial Sequence Antisense Oligonucleotide 11 tggcatgatc tggtactccc 20 12 20 DNA Artificial Sequence Antisense Oligonucleotide 12 ccacgtcagg aatattttct 20 13 20 DNA Artificial Sequence Antisense Oligonucleotide 13 tctctctctc tattggagtc 20 14 20 DNA Artificial Sequence Antisense Oligonucleotide 14 aagcgatgct gccaggttca 20 15 20 DNA Artificial Sequence Antisense Oligonucleotide 15 gaaaattctt tggaatcttc 20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 agctgatctc atgaaaattc 20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17 tcataatctt ttcatgaaat 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18 tctgagatca taatcttttc 20 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19 cctctttacg ctttcatgta 20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 ccctgcccca gagatttgtg 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 gcggtgccca tctcagccct 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 cccagatccg cggtgcccat 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 ttagttggtg taggcactga 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24 acattagttg gtgtaggcac 20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 tgttatagga ttcaattgta 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 atacgatagg gttcatgtta 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 gtactcccaa tatacgatag 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 ctggtactcc caatatacga 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29 gatctggtac tcccaatata 20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 ggacctgtgg catgatctgg 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 tggatcacca acatgatcag 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 ccctggcttt aactctgacc 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 ccaaccctgg ctttaactct 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34 tgtccaaccc tggctttaac 20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 actttgcata ggcagattct 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 ctgactttgc ataggcagat 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 tacagcaaat tcttctgact 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 cagtttaggt ggtccaattt 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39 ctgatatcca gtttaggtgg 20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 catgatttgc ttctcctcct 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 gtctccattt acaaaaactg 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 ttcctgctcg tctccattta 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 aatcgacttc ctgctcgtct 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44 atgtaacagg tagtttcggg 20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 ctcacataca cattgtacac 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 tcattctcac atacacattg 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 ttccgttcat tctcacatac 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48 gcgtgagtat tttatactgg 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 ctgcgtgagt attttatact 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 acaatcatct tccttctgcg 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 tcacaatcat cttccttctg 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 cactgaatct cgtcacaatc 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53 actggcactg aatctcgtca 20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 tactgagaat tcagtgagga 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 cagtactgag aattcagtga 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 aaacacagta ctgagaattc 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 cttctgctga aacacagtac 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58 ccccacacat gtaagactcc 20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 aacaccccac acatgtaaga 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 cttttatact gctattgaaa 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 gcagcaacaa ctggaatcca 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 gtagtaaagc agcaacaact 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63 ctaagcacta gaaagagtag 20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 taaagtagca cttcttacca 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 ggttttgtct ctaaagtagc 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 atggctggta tgacgtgatg 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 gttgctggag acaacggctc 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68 aactgttgct ggagacaacg 20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 gttccacttt tcctggattg 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 agttcttctg tatgttccac 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 aaagttcttc tgtatgttcc 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 gatggctgcc cgggaccacg 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73 tcagatggct gcccgggacc 20 74 20 DNA Artificial Sequence Antisense Oligonucleotide 74 gaagaactct ctctctctat 20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 ctacttaaag gtgaagaact 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 actggttact acttaaaggt 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 gagtgatacg agtttaaagc 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78 gagtgatcac tctcagaaca 20 79 20 DNA Artificial Sequence Antisense Oligonucleotide 79 tctggagtga tcactctcag 20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 gctatgtgat tccagacagc 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 ggaaattctg agtcagataa 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 ttacggttat gagctcttgt 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83 ttatcataac caaaggaggt 20 84 20 DNA Artificial Sequence Antisense Oligonucleotide 84 tatcatccac aagtagatcc 20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 ccgctatcat ccacaagtag 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 ctctttaccg ctatcatcca 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 aaggactctt taccgctatc 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88 cttctgttgg tctataacca 20 

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targeted to a nucleic acid molecule encoding Interferon gamma receptor 1, wherein said compound specifically hybridizes with and inhibits the expression of Interferon gamma receptor
 1. 2. The compound of claim 1 which is an antisense oligonucleotide.
 3. The compound of claim 2 wherein the antisense oligonucleotide has a sequence comprising SEQ ID NO: 11, 12, 13, 16, 17, 18, 22, 24, 25, 28, 29, 30, 41, 44, 45, 48, 53, 55, 57, 59, 61, 63, 65, 66, 67, 68, 70, 71, 73, 77, 79, 86 or
 87. 4. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.
 5. The compound of claim 4 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 6. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
 7. The compound of claim 6 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.
 9. The compound of claim 8 wherein the modified nucleobase is a 5-methylcytosine.
 10. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.
 11. A compound 8 to 50 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of an active site on a nucleic acid molecule encoding Interferon gamma receptor
 1. 12. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
 13. The composition of claim 12 further comprising a colloidal dispersion system.
 14. The composition of claim 12 wherein the compound is an antisense oligonucleotide.
 15. A method of inhibiting the expression of Interferon gamma receptor 1 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of Interferon gamma receptor 1 is inhibited.
 16. A method of treating an animal having a disease or condition associated with Interferon gamma receptor 1 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of Interferon gamma receptor 1 is inhibited.
 17. The method of claim 16 wherein the disease or condition is an autoimmune disorder.
 18. The method of claim 17 wherein the autoimmune disorder is autoimmune thyroiditis, diabetes, multiple sclerosis, autoimmune arthritis, autoimmune insulinitis or Crohn's disease.
 19. The method of claim 16 wherein the disease or condition is cancer.
 20. The method of claim 16 wherein the disease or condition is caused by aberrant apoptosis. 